Ch 12. Researchers can insert desired genes into plasmids, creating recombinant DNA and insert those plasmids into bacteria Bacterium Bacterial chromosome.

Ch 12

Researchers can insert desired genes into plasmids, creating recombinant DNA and insert those plasmids into bacteria

Bacterium

Bacterialchromosome

Plasmid

1 Plasmidisolated

3 Gene insertedinto plasmid

2 DNAisolated

Cell containing geneof interest

DNAGene ofinterest

Recombinant DNA(plasmid)

4 Plasmid put intobacterial cell

Recombinantbacterium

5 Cell multiplies withgene of interest

Copies of proteinCopies of gene

Clone of cellsGene for pestresistanceinserted intoplants

Gene used to alter bacteriafor cleaning up toxic waste

Protein used to dissolve bloodclots in heart attack therapy

Protein used tomake snow format highertemperature

Figure 12.1

Creating recombinant DNA using restriction enzymes and DNA ligase

Restriction enzymerecognition sequence

G A A T T CC T T A A GDNA1

2

3

4

C T T A AA AT TC

A AT TCG

C T T A AAddition of a DNAfragment fromanother source

Two (or more)fragments sticktogether bybase-pairing

G A AT T CC T TA A G

G A AT T CC T TA A G

5

DNA ligasepastes the strand

Restriction enzymecuts the DNA intofragments

Recombinant DNA molecule

GG

Sticky end

G

Figure 12.2

Cloning a gene in a bacterial plasmid

1Isolate DNAfrom two sources

2Cut both DNAswith the samerestriction enzyme

E.coli

PlasmidDNA

Gene V

Sticky ends

3 Mix the DNAs;they join bybase-pairing

4 Add DNA ligaseto bond the DNA covalently

5 Put plasmid into bacteriumby transformation

Gene VRecombinant DNAplasmid

Recombinant bacterium

6 Clone the bacterium

Bacterial clone carrying manycopies of the human gene

Human cell

Figure 12.3

Cell nucleus

Isolation of mRNAand addition of reversetranscriptase; synthesisof DNA strand

RNA splicing2

Transcription1

3

Breakdown of RNA4

Synthesis of secondDNA strand

5

mRNA

DNA ofeukaryoticgene

IntronExon

RNAtranscript

Exon Intron Exon

Reverse transcriptaseTest tube

cDNA strandbeing synthesized

cDNA of gene(no introns)

Reverse transcriptase makes single stranded DNA from mRNA

cDNA

Recombinant cells and organisms can mass-produce gene products

Table 12.6

Therapeutic hormones & vaccines– In 1982, humulin, human insulin produced by

bacteria• Became the first recombinant drug approved by the

Food and Drug Administration

– Vaccines are harmless mutants or derivative of a pathogen that stimulate the immune system

Figure 12.7A

• GM organisms– Acquired genes by artificial means– Transgenic organisms

Agrobacterium tumefaciens

DNA containinggene for desired trait

Tiplasmid

Insertion of geneinto plasmid

RecombinantTi plasmid

1

Restriction site

Plant cell

Introductioninto plantcells

2

DNA carrying new gene

Regenerationof plant

3

Plant with new trait

Gene therapy (or the alteration of an afflicted individual’s genes) may someday help treat a variety of diseases

Cloned gene(normal allele) 1 Insert normal gene

into virus

2 Infect bone marrowcell with virus

3 Viral DNA insertsinto chromosome

4 Inject cellsinto patient

Bonemarrow

Bone marrowcell from patient

Viral nucleicacid

Retrovirus

Figure 12.13

• DNA profiling

Crime sceneDNA isolated1

Suspect 1 Suspect 2

DNA of selectedmarkers amplified

2

Amplified DNA compared

3

Cycle 1yields 2 molecules

21 3

GenomicDNA

Cycle 3yields 8 molecules

Cycle 2yields 4 molecules

3 5 3 5 3 5

Targetsequence

Heat toseparateDNA strands

Cool to allowprimers to formhydrogen bondswith ends oftarget sequences

35

3 5

35

35 35

Primer New DNA

5

DNApolymerase addsnucleotidesto the 3 endof each primer

5

PCR used to amplify target section of DNA

Gel electrophoresis sorts DNA molecules by size

+ +

– –

Powersource

Gel

Mixture of DNAmolecules ofdifferent sizes

Longermolecules

Shortermolecules

Completed gel

Figure 12.10

– After digestion by restriction enzymes the fragments are run through a gel

–

+

Longerfragments

Shorterfragments

x

w

y

z

y

1 2

Figure 12.11B

DNA fingerprinting can help solve crimes

Defendant’sblood

Blood fromdefendant’s clothes Victim’s

blood

Figure 12.12A Figure 12.12B

• STR– Short sequences of DNA repeated many

times in a row– STR analysis compared lengths of STR

sequences at specific sites on the genome

• Used in forensic investigations

STR site 1

Crime scene DNA

STR site 2

Suspect’s DNA

Number of short tandemrepeats match

Number of short tandemrepeats do not match

How Restriction Fragments Reflect DNA Sequence– Restriction fragment length polymorphisms (RFLPs)

reflect differences in the sequences of DNA samplesCrime scene Suspect

w

x

y y

z

CutCut

Cut

DNA from chromosomes

C

C

G

G

G

G

C

C

A

C

G

G

T

G

C

C

C

C

G

G

G

G

C

C

C

C

G

G

G

G

C

C

Figure 12.11A